KR20000002473A - Medication for preventing and curing degenerative cerebral neural disease - Google Patents

Abstract

PURPOSE: A medication for preventing and curing a degenerative cerebral neural disease such as a dementia senilis is provided to improve an energy metabolism and to postpone a progress of aging. CONSTITUTION: A material for preventing and curing a dementia senilis comprises: 1.6-2.4 wt% of ginseng; 0.7-1.3 wt% of astragalus; 0.7-1.3 wt% of white atractylis; 0.7-1.3 wt% of white paeonia; 0.7-1.3 wt% of Korean angelica root; 0.7-1.3 wt% of cnidium; 0.7-1.3 wt% of corium; 0.7-1.3 wt% of liquorice; 0.7-1.3 wt% of wilfordi root; and 0.7-1.3 wt% of broiler.

Description

Prevention and treatment of degenerative cerebral nervous system disease

The present invention relates to a prophylactic and therapeutic agent for degenerative cerebral nervous system diseases such as senile dementia, vascular dementia, and Alzheimer's dementia.

In observing humans from the point of view of oriental medicine, many diseases of the human body are attributed to the disharmony of mind and body. Jeong, qi, god, blood (精, 氣, 神, 血) in terms of jeong, qi, god (精, 氣, 神) is an important basic component of the human body, jeong (精) is a unit of formation, Ki (氣) is the life energy of human physiological and external activities, deity is the spirit as the purification of life, and blood is regarded as the concept of biological material encompassing blood. have. The corresponding ideological theory is the Dead Sea Theory, where the Dead Sea refers to Qin, Ancient, Yu, and Liquid. In other words, the Dead Sea Theory is the four major nutrients in which the "Sagi" of Sugok refers to Jin, Go, Yu, and Liquid. It is a theory that explains the cycle and its main functions. When the temperature, quantity, heat, heat, heat, heat, and heat of grains start Yanbian in the body, in the first stage, Jin, Go, Yu (油), four major nutrients and liquids ("quasi sea"), namely Jinhae, Gohae, Hazardous, and Liquid Sea (Juhai, 膏 海, 油 海, 液 海) to the second stage Into God, Ki, blood, Jeong (神, 氣, 血, 精) into four major blood substances (四大 氣血 物質), "Fusa Sea (後 四海), that is, the Ni Sea, the Mak Sea (膜 海), blood sea ( It is believed that it maintains the normal activities of each organ through the sea and the sea. Thus, in oriental medicine theory or ideological theory, it is believed that the function of deity can be maintained by protecting jeong, qi, blood (精, 氣, 혈). From the point of view of Western medicine, blood and blood can be understood as corresponding energy sources and processes of energy metabolism (production), respectively. Therefore, the brain's nervous system can be considered as a god, and it can be considered that energy and metabolic function of the god can be maintained by helping Gi and blood.

Sasang disease is divided into two types: the surface disease and the disease, and the disease results in the blood, muscle, meat, bone, and bone of the human body. This disease causes consumption and dysfunction (이) causes disorders in nutrient metabolism (病症), namely the gastrointestinal (esophageal region), stomach, small intestine, and bowel disorders. The concept of aging or dementia is regarded as a disease that appears as a result of the progression of epidemic and disease in ideology.

In the past, the noise of Jeopjeondaebo-tang (Su-Jin-Dae-Bon-Tang) is one of Sasang's prescriptions, as described in Dong Bo-gam (Ho Jun), Dong-Suse Suwon (Ijema), and Taepyeong Hyemin-hwa National Defense (Jinsamun). It has been used in the etiology of the pathopathy, specifically, the drug's effect is to enhance the expression of the noise person (the innate resistance of the noise person) and corrective ganglia (補 精 强 筋骨), degenerative arthritis, infertility, menstrual disorders, dysmenorrhea, tonsils, Only known to apply to the symptoms of skin diseases, uterine disease. The inventors of the present invention intend to use it for the treatment and prevention of degenerative cerebral nervous system disease by applying the noise of Junction.

As aging progresses, the brain develops severe atrophy. The most atrophic part is the cerebrum, which is known to show significant atrophy in Alzheimer's dementia patients. Atrophy of the cerebral cortex in the atrophy of the brain due to aging can be considered to be due to a decrease in surface area, and atrophy of the cerebral cortex by Alzheimer's type dementia is known to be due to a decrease in both surface area and thickness.

As the brain contracts with aging, energy metabolism decreases. In the cerebral cortex of senile dementia patients, glucose metabolism is known to decrease by 50-70%. Glucose, the main source of energy in the brain, has several stages of biochemical metabolic processes, leading to acetylcholine (Ach: acetylcholine), glutamine (glutamate), aspartate, and gamma aminobutyric acid (GABA). ), Glycine (glycine), adenosine triphosphate (ATP) and acetyl CoA (acetyl CoA) to produce such a neurotransmitter and energy source necessary to maintain the neuronal physiological function. Among the various functions of the cerebral function, cognitive and memory functions are directly related to intracellular cholinergic activity, and cholinergic deficit observed in senile dementia is a result of glucose metabolism disorder. Glucose turnover and decreased ATP production are known as specific symptoms of dementia brain disease. Therefore, cholinergic deficit, neuronal atrophy, amyloid deposition, etc. in dementia brain disease can be considered as a secondary phenomenon due to the degradation of energy metabolism.

On the other hand, there is a theory in aging that claims to be due to protein synthesis and dysfunction. In this regard, senescent cells, including nervous system cells, accumulate abnormally functioning proteins, one of which is methylated protein. Related to the functional recovery of these modified proteins is protein methylase II (PM II), which is known to play an important role in the functional recovery of proteins related to aging.

There are a number of factors that lead to death of cerebral nervous system cells, but in the cellular morphology the path of death can be divided into apoptosis and necrosis. They can be observed with different morphological features, but in general, apoptosis is an active and physiological process and necrosis is known as a passive and pathological response. However, one thing that can be found in these two different processes is that they cause a decrease in cell viability.

Figure 1a to 1d is a photograph showing the effect of the composition of the present invention on the apoptosis (apoptosis) of primary cultured cerebral cortical cells induced by glutamine;

2a to 2d are photographs showing the effect of Angelica peony peony on apoptosis of primary cultured cerebral cortical cells induced by glutamine;

3 is a photograph showing the effect of the composition of the present invention on chromosomal DNA fragmentation of glutamine-induced cortical neurons;

4A to 4F are photographs showing growth of primary cultured cerebral cortical cells without addition of growth factors;

5a to 5c are photographs showing the effect of the composition of the present invention and Angelica peony mountain on the spontaneous degeneration of cultured neurons;

6a to 6f are photographs showing the effect of the composition of the present invention on the spontaneous degeneration of neurons cultured in primary culture by double fluorescence staining;

7 shows body weight changes of SAM R1 mice treated with the present compositions;

8 is a diagram showing the weight change of SAM P8 mice administered the composition of the present invention;

9 is a view showing changes in the amount of ATP in the cerebral cortex of SAM R1 mice and SAM P8 mice administered the composition of the present invention,

10A to 10F are photographs showing pathological changes in the brain of SAM P8 mice to which the composition of the present invention was administered.

The composition and the ratio of the composition of the present invention are summarized in Table 1. An adult dose (60 kg) is suitable for 45g.

Collection, drying, storage of medicines and the law of the medicine (法製) is different depending on the medicine, it is in the usual way.

Table 1. Medicinal composition of the present invention composition

Pharmaceutical weight ratio Ginseng 1.6-2.4 Yellow 0.7-1.3 White 0.7-1.3 White 0.7-1.3 Ears 0.7-1.3 Cheong 0.7-1.3 True Blood 0.7-1.3 Persimmon 0.7-1.3 White sewage 0.7-1.3 Broiler 0.7-1.3

Sanctions can be prepared with a hot water for 1 hour with purified water 10 times the total weight of the medicine. It can also be prepared as an extract. For 10 doses (450 g), place in a 5,000 ml round flask, add 3,000 ml of purified water, attach a cooler, heat and warm for 2 hours, and filter the filtrate. It was concentrated under reduced pressure in a rotary evaporator (rotary evaporator) and then completely dried in a 40 ° C. decompression dryer to obtain an extract 110.5 g, or 3 hours of heating and heating to obtain an extract 90.0 g. According to the amount of medicine to be prepared by the extract is adjusted the amount of the purified water and the heating hot water. In the form of medicines, a single dose of the medicated liquid and extract may be prepared as a drink.

In addition, the medicinal herbs can be washed with purified water and dried to prepare a powder (powder). The form of the medicament may be in powder form, or may be prepared in capsules, granules, tablets and dragees.

As another method, the powder may be made into a round shape by kneading with a paste or honey after making the powder. It may also be prepared from other crystals.

A single dose of this type of medicine is administered orally three times a day. The duration of administration is adjusted according to the patient's symptoms.

Administration of the composition of the present invention exhibits known efficacy and preventive and therapeutic effects of other degenerative cerebral nervous system diseases such as senile dementia, vascular dementia, and Alzheimer's dementia.

In Vitro experiments using primary cultured neurons and in vivo experiments using disease model animals to demonstrate the efficacy of the medicinal herbs for degenerative cerebral nervous system diseases are as follows.

First, look at the in vitro experiment method.

From the point of view of oriental medicine and western medicine, aging is considered to be a degeneration of naturally occurring physiological functions, and neural cells die naturally. At the cellular level, this phenomenon can be thought to be caused by apoptosis and / or necrosis.

First, the effects of herbal medicine on apoptosis induced by glutamine administration (0.7-1.0 mM) and lethality of naturally occurring cells were investigated.

Second, as a characteristic change in apoptosis, the formation of chromosomal DNA fragments of about 180 bp is known by activation of DNA endonuclease, and thus induced by glutamine by administration of the composition of the present invention. In order to observe the cellular protective action against apoptosis of the neuronal cells, DNA fragmentation was observed.

Third, the delayed (improvement) effect of the composition of the present invention was observed for spontaneous degeneration of cultured cerebral cortical nervous system cells.

Fourth, the delayed (improvement) effect of the composition of the present invention on the natural regression of neurons using double fluorescence staining in terms of specific morphological changes including apoptosis and necrosis. Was observed.

Fifth, lactate dehydrogenase (LDH) in the cytoplasm is discharged out of the cell membrane when there is damage to the cell membrane by necrosis or cytotoxic substances. Therefore, the amount of LDH contained in the culture was measured in order to search for the efficacy of medicinal herbs on the natural deterioration (aging) of cell function and to evaluate the damage state of the cell membrane.

In this observation, Angelica peony acid known to improve glutamine-induced apoptosis was used as a positive control medicine.

On the other hand, the cerebral cortex separation method and cell culture method for obtaining common nervous system cells in an in vitro experiment are as follows.

Deformed cerebral cortex of mice 1-2 days old to make a block of about 0.5 mm ³ , and then modified artificial cerebrospinal fluid (ACSF) containing 0.1% BSA and 0.025% trypsin to separate neural tissue into single cells. : modified artificial cerebrospinal fluid, 136mM NaCl, 5.4mM KCl, 0.4mM KH ₂ PO ₄ , 0.5mM MgCl ₂ , 0.4mM MgSO ₄ , 0.3mM Na ₂ HPO ₄ , 15mM tri (hydroxymethyl) aminomethane, pH 7.4) At 37 ^° C. for 15 minutes. In order to increase the purity of the isolated single cells, unfractionated tissue sections were precipitated and removed with an ACSF solution containing no BSA and trypsin. This washing process was repeated three times and the cell number was adjusted to 10 ⁶ / ml to dispense 2ml into a 35mm culture plate. The isolated cerebral cortex was DMEM (Dulbecco's modified Eagle culture solution containing 95% air / 5% CO _2, 10% fetal calf serum, 100 U / ml penicillin, and 100 μg / ml streptomycin under humidified conditions). , Glutamine was not added), and cell culture was performed. In order to use the conditions in which the degeneration of the nervous system cells occurs quickly, it was performed under the condition that no growth factor was added, and the incubation time was adjusted according to the purpose of the experiment. In addition, in order to increase the number of neurons, 5 μM cytosine arabinoside was added to the culture solution on the 2nd day of cell culture, and then replaced with normal medium for 24 hours.

Next, look at the in vivo experimental method.

As a disease model animal, a 24-week-old patient with clinical and pathological features such as learning and memory impairment in dementia, spongiform degenerative lesions, PAS staining granule structure, and β / A4 proteinaceous granule structure SAM P8 mice (congenital aging promoting mice) and 24 week old SAM R1 were selected as normal animal models. Pre-heated liquid is prepared by applying heat for 1 hour with water 10 times the total weight of the medicine of Table 1. Dosage is determined according to the ratio of oral dose to mouse body weight (mouse: 2 ml / Kg), wherein three times the oral dose, the clinical use dose, is administered orally six times a week for 12 weeks.

First, the body weight and brain weight were changed after the administration.

Second, changes in cellular energy metabolism are closely related to the intake of energy sources from outside and the energy production capacity of cells. Therefore, the change of blood glucose concentration was evaluated to prove more specific result.

Third, the supply of the necessary amount of energy source required for the production of energy required for cell survival is very important in maintaining the homeostasis of the cell, the blood glucose and tissue substrate utilization can be considered as a criterion for evaluating this. Therefore, for this purpose, we observed the blood glucose level in the cerebral cortex as well as the above-mentioned blood glucose measurement in animals treated with Goyang image tang.

Fourth, in order to investigate the effect of the composition of the present invention on the energy metabolism of the cerebral cortex, the cerebral cortex was separated and the intracellular ATP content was measured. Energy metabolism is an essential process for the maintenance of physiological function in all cells, including nervous system cells, and the reduction of energy metabolism is well known in the degenerative lesions of neurological cells that represent dementia. This is because it is also considered as the primary etiology for the formation of lesions.

Fifth, the change in PM II activity on SAM P8 (congenital aging promoting mouse) was investigated and the effect of the composition of the present invention was observed. The background is as follows. PM II is involved in the recovery or removal of abnormal proteins in cells by aging and is known to be present in high concentrations in mammalian brain tissue. In Alzheimer's disease or Alzheimer's type dementia, there are known etiologies in which abnormal proteins such as intracellular neurofibrillary tangle and extracellular amyloid plaque are deposited. As described above, the protein plays a pivotal role in the expression of cellular functions, as well as the pathologies and biochemical changes occurring in aging and dementia neurons. Although there is no report on the relationship, pathology, and biochemical significance of protein methylation in SAM model animals, it is secondary if the composition of the present invention has an effect on maintaining physiological function according to improvement of cellular energy metabolism. It can be considered that this may also affect the function (form) of the protein.

Sixth, the degenerative lesions observed in the brain of patients with dementia and brain tissues of SAM P8 were compared, and at the same time, histopathological examination was performed to investigate the effect of the composition of the present invention on the degeneration of brain neuronal cells. . The experimental background is as follows. Three important pathological morphological findings of SAM P8 were mainly sponge sponge degeneration in the brainstem region, formation of PAS-positive granules in the hippocampus and β / A4 protein granules (β / A4). protein-like immuno-reactive granules are formed, with pathological findings similar to those of human senile, Alzheimer's dementia.

The present invention will be described in detail by the following examples.

<Example 1>

Improvement of apoptosis induced by glutamine with the composition of the present invention

After 16 to 24 hours administration of glutamine, the compositions of the invention (0.01, 0.1 mg / ml) were administered to cultured nervous system cells. FIG. 1A shows only 1 mM glutamine, FIG. 1B shows only 1 mM glutathione, and FIG. 1C shows 1 mM glutamine + 0.1 mg / ml of zonazitanbotang, and FIG. 1D shows 1 mM glutamine + 1.0 mg / ml of zonazibotan Result (glutamine is administered 16-24 hours). As a result, it can be seen that the degeneration of neuronal neurons induced by glutamine is significantly reduced. In particular, the denaturation of neurites due to glutamine was significantly reduced. The results for the composition of the present invention were very similar to the efficacy of the glycojaxak acid of Comparative Example 1 below.

Comparative Example 1

For cultured nervous system cells, Angelica sacrum is known to improve apoptosis by glutamine. Therefore, Donggui Pakjaksan was used as a positive control medicine for the composition of the present invention. Figure 2b is not administered glutamine, Figure 2a is only 1mM glutamine, Figure 2c is 1mM glutamine + 0.1mg / ml glycomic pelican acid, Figure 2d is 1mM glutamine + 1.0mg / ml of Dongguk Paksan Results of dosing (glutamine is administered 16-24 hours). The results showed that Dangwi Paksan significantly improved the degeneration (degeneration) of neurons and neurites by the administration of glutamine.

<Example 2>

DNA fragmentation aspect

In the observation method, the mouse cerebral cortical nervous system cells were first administered for 2 days after the culture of the present invention at a concentration of 0.1, 1.0 mg / ml from the 5th day from the culture, and then 0.7 mM glutamine was administered for 24 hours. Cells were then separated and 300 μl digestion buffer (100 mM NaCl, 25 mM EDTA, 0.5% sodium dodesylsulfate, 10 mM Tris / HCl, 0.1 mg / ml protenase K, pH 8.0) was added to the 35 mm plate to separate DNA. It was. Precipitating genomic DNA with isoamyl alcohol / methanol / chloroform (25: 24: 1), 70% ethanol and 100% ethanol, and then separating the DNA fractions by electrophoresis on a 1.5% agarose gel. The DNA properties were observed by ethidium bromide staining.

As shown in FIG. 3, many DNA fragments of 180-200 bp size could be observed by glutamine administration. This change was found to be significantly suppressed by the administration of the composition of the present invention. This is consistent with the inhibitory effect of Angelica peony acid on chromosomal DNA fragmentation by known glutamine.

<Example 3>

-Effects on spontaneous degeneration of neurons

FIG. 4 shows the results of cell growth in DMEM culture medium without insulin, transferrin, and selenium (FIGS. 4A to 4F show 2, 4, 6, 8, 10, Observed on day 13). Newlite showed active growth from 7 to 8 days, with the best growth around 8 days. After 10 days after the start of the degeneration showed a sharp degeneration of only axon (axon) cell morphology was observed around 11 days, the appearance of the cell was able to clearly determine the degeneration of the cells. Therefore, in order to investigate the effects of Angelica peony peony and the composition of the present invention as a positive control medicine against spontaneous degeneration of cultured nervous system cells, each medicine was added to the culture medium at a concentration of 1 mg / ml for 7 to 11 days of culture. The effect on developmental degeneration was investigated. Figure 5 is the result observed on the culture day 11, the end of the experiment. In the control group not administered the drug (FIG. 5a), remarkable degeneration of neuronal cells such as neurons and neurons was observed.

Degeneration of these cells was significantly improved by administration of the composition of the present invention (FIG. 5C). The degree of delay (improvement) on the natural regression of these cells was found to be greater than that of the present invention composition, Angelica peony peony (Fig. 5b), which is a positive control drug, and these results were cortical energy observed in Example 8 below. Very consistent with the results for production capacity.

<Example 4>

Effect on the Morphological Changes of Naturally Degenerative Neurons by Dual Fluorescence Staining

The double fluorescence staining method used was as follows. Fluorescence staining using acridine orange and ethidium bromide was used to distinguish the relationship between apoptosis and necrosis on cell activity. Cortical neuronal cells were treated with the composition of the present invention at a concentration of 0.1 mg / ml for 6-14 days after the start of the culture. Cultures were removed and collected to measure lactic acid dehydrogenase (LDH), and then washed three times with phosphate buffered celine (PBS). Acridine orange and ethidium bromide were adjusted to a concentration of 2 μl / ml and observed for 2-3 minutes at room temperature. Acridine orange staining is determined by the extent of cell membrane penetration of fluorescent DNA-binding staining reagents for viable and non-viable cells for a certain number of cells. Therefore, acridine orange staining can assess whether the cells have undergone apoptosis and the degree of apoptosis (early or late). Acridine orange is bound to and intercalated with DNA, but can only be bound to RNA, dyes bound to DNA appear green, and reagents bound to RNA are red-orange. It will show color. Itidinium bromide will enter the cell only if there is an abnormality in the cell membrane (eg dead cells). Thus, in dead cells, the nucleus is bright orange and the cytoplasm is dark red. On the other hand, in the dead cells, the nucleus of normal or apoptosis appears to be bright orange. From these differences in fluorescence, early and late apoptosis (apoptosis) and necrosis (necrosis) can be distinguished.

6a shows a control group, FIG. 6c shows a 0.1 mg / ml glycomic acid, 6e shows a 0.1 mg / ml administration of the present invention, and FIG. 6b shows a control group, FIG. 6d. Is the result of the administration of 0.1 mg / ml glycomic acid, 6f was administered 0.1 mg / ml of the composition of the present invention, respectively, by double fluorescence staining (attached color photo as an attachment).

As a result, as shown in FIG. 6a, neuronal sheep cells showed significant regression at 14 days of culture, and only the exon remained. When stained by double fluorescence method, many neuronal cells including neurons showed orange color and staining of fragments was observed. This clearly demonstrates that many cells are in the state of late apoptosis or necrosis on day 14 of culture. On the other hand, 0.1 mg / ml of the present invention composition and glycozac acid were administered to the culture solution for 9 days over 6 to 14 days of culture, and cell degeneration was significantly improved (FIGS. 6E and 6C). Furthermore, when staining by double fluorescence was applied to these cells, many neuronal cells including neurons were stained with blue staining and fragments, unlike the control group not administered the composition of the present invention. ) (Figures 6f, 6d). This demonstrates that a large number of cells are normal or in early apoptotic state, unlike cells that have not been administered compositions, etc. of the present invention. That is, in cultured nervous system cells, the Angelica sacrum and the composition of the present invention can be considered to have an effect of delaying cell degeneration due to apoptosis or necrosis.

Example 5

Free of lactate dehydrogenase (LDH)

LDH is a tetrameric protein and has a function related to the conversion to lactate or pyruvate by oxidation / reduction reactions. While pyruvate is reduced, oxidation of reduced nicotineamide-adenine dinucleotide (NADH) at the same mol concentration occurs to form oxidized nicotinamide-adenin dinucleotide (NAD ⁺⁾ . Depending on the degree of oxidation of NADH, the absorbance at 340 nm decreases, which is proportional to the amount of LDH contained in the sample.

Table 2. Free LDH Volume for Each Prescription

Prescription mg / ml Mean ± Standard Deviation Importance Control 31.6 ± 1.13 glycozac acid 0.1 20.6 ± 0.80 ** 1.0 21.7 ± 1.06 ** composition of the present invention 0.1 16.7 ± 2.33 ** 1.0 21.5 ± 0.27 **

** indicates significant difference p <0.01 for control.

Table 2 shows the amount of LDH released into the culture solution during the last 1 day of the experiment divided into the group treated with the composition of the present invention and the untreated group for 6 to 9 days of culture. When 0.1 to 1.0 mg / ml of the composition of the present invention was added to the culture solution, a significant difference between the untreated control group and the LDH was observed. It was shown to have higher efficacy than Dangwi Paksan (0.1-1.0 mg / ml), a positive control drug that showed a significant difference (p <0.01) compared to the amount of LDH observed in the non-administered control group. These results are in good agreement with the results obtained from morphological observation (FIG. 6), demonstrating that the composition of the present invention has the effect of delaying and inhibiting spontaneous degeneration of cultured cells.

<Example 6>

Changes in body weight and brain weight

Body weights were measured at the beginning of the experiment and then weekly to change the dosage of the medicinal herbs according to the change in body weight of each animal. In addition, clinical observations of all animals in the administration group and the no-administration group were conducted six times a week to investigate the health status of all animals. At the end of the experiment, animals were weighed, anesthetized with ethyl ether, and brains were removed to remove water, and then brain weights were measured to evaluate the relative weight to weight ratio. Nine SAM R1 mice and 18 SAM P8 mice were used.

The results are as follows.

By administration of the composition of the present invention at a clinical dose for 12 weeks, the animals treated in the administration showed a significant increase in activity compared to the non-administered control animals throughout the experimental period. This difference was more pronounced in SAM P8 than in SAM R1. As shown in FIGS. 7 and 8, the body weight of the 36-week-old SAM P8 mouse was lower than that of the control animal, SAM R1. When the composition of the present invention was orally administered to SAM R1 (FIG. 7) and SAM P8 (FIG. 8) for 12 weeks, body weight began to be changed after 1 week of administration, and at 12 weeks after the end of the experiment, body weight was compared with that of no-administration control animals. An increase in was observed. However, the rate of change in weight gain was more pronounced in SAM P8 than in SAM R1. Differences in clinical observations and weight gain rates between normal animals (SAM R1) and abnormal animals (SAM P8) by administration of the composition of the present invention are attributable to the effect of herbal medicine to maintain homeostasis of body function, which is the basic theory of herbal medicine. On the other hand, atrophy of brain tissue with aging progress is known to cause abnormal physiological functions.

Table 3. SAM brain weight change

Animal Animals Brain Weight (g) Weight (g) Brain / Weight (x100) SAM R1 Control 9 0.45 ± 0.005 33.9 ± 1.73 1.34 ± 0.07 Experiment 9 0.46 ± 0.004 ^* 34.5 ± 0.94 1.35 ± 0.04 SAM P8 Control 18 0.45 ± 0.003 32.1 ± 1.04 1.41 ± 0.04 Experiment 18 0.48 ± 0.004 ^** 33.4 ± 0.98 1.44 ± 0.03

* Means significant difference p <0.05 and ** means significant difference p <0.01 for the control group.

The above values and the values given in the examples below represent mean ± standard deviations.

Table 3 shows the changes in brain weight of 36-week-old SAM R1 and SAM P8 by administration of the composition of the present invention. No difference in brain weight was observed between SAM R1 and SAM P8 without administration of the composition of the present invention. However, after oral administration of the composition of the present invention for 12 weeks, an increase in brain weight in SAM R1 and SAM P8 was observed (p <0.05 or p <0.01). This increase in brain weight was more pronounced in SAM P8 than in SAM R1, as seen by weight change. And when the brain weight is converted to the relative weight with respect to the weight of the animal subject, high values were seen in the composition-administered group of the present invention.

<Example 7>

Changes in Glucose Content in the Blood

After anesthesia with Ethyl ether, the animal's abdominal cavity was opened and blood was collected through the upper abdominal vein. After allowed to stand for clotting blood to room temperature, at 4 ^o C to remove the serum by centrifugation of 8000rpm, 10 min and stored frozen at -70 ^o C until measurement. Glucose was measured for absorbance at 340 nm by hexokinase / glucose-6-phosphate dehydrogenase.

Table 4 summarizes the measurement results and shows the change in blood glucose concentrations of SAM R1 and SAM P8 at 13 weeks after the administration of the present composition. Serum glucose concentrations between SAM R1 and SAM P8 without medicinal herbs were found to be somewhat higher in SAM R1, but no statistical significance was found between the two groups. The blood glucose concentrations of SAM R1 and SAM P8 administered the present invention showed lower results than the untreated control group. The slightly higher blood glucose concentration of SAM R1 in SAM P8 may be due to an increase in feed intake associated with increased activity, but the decrease in blood glucose by administration of the composition of the present invention may be due to an increase in glucose utilization. .

Table 4. Blood Glucose Content

Per animal animal condition (mg / dl) SAM R1 (9) Control 211.8 ± 15.85 SAM R1 (9) Experimental 200.4 ± 5.91 SAM P8 (9) Control 198.5 ± 10.83 SAM P8 (16) Experimental 190.4 ± 8.32

<Example 8>

Changes in Cellular Glucose Content and ATP Content

The measurement method of the intracellular glucose content is as follows. For treatment of the cerebral cortex isolated from SAM mice, 600 μl of phosphate buffered saline containing 0.1% Triton X-100 was used as the buffer. The grinding of the tissues was done uniformly for all samples by 20 repetitions in an ice-cold state using a glass-teflon homogenizer (for 1 ml). Centrifugation was performed at 8,000 rpm for 10 minutes to separate glucose and tissue sections freed from buffer. After centrifugation, the supernatant was taken and used for the measurement of glucose and protein. Glucose was measured by the same method as blood glucose. The glucose content of each sample was expressed in terms of the ratio of the measured protein amount of the same sample.

On the other hand, the method of measuring the ATP content is as follows. At the end of the administration, cerebral cortical tissues of SAM R1 and SAM P8 were ground with 1 mM ethylenediaminetetraacetic acid (EDTA) and 10% trichloroacetic acid (TCA) to release intracellular ATP into the buffer. Free ATP was centrifuged at 15,000 rpm for 10 minutes to separate tissue debris. The supernatant was taken and used as a sample for measuring the protein and ATP content. The measurement of ATP was performed by the chemiluminescence method which slightly improved the method of Jung et al. The amount of light produced in proportion to the amount of ATP contained in the sample was measured by a luminometer. The number of animals used was 9 and 18 SAM R1 and SAM P8, respectively.

Figure 9 shows the results of the measurement of intracellular ATP content, each column shows the mean ± standard deviation, ** indicates a significant difference p <0.01 for the control. The intracellular ATP contents of 36-week-old SAM R1 and SAM P8 were different. The ATP content of the SAM P8 cerebral cortex was somewhat lower than that of SAM R1. However, no statistical significance was found between the two groups. 12 weeks of administration of the composition of the present invention compared to animals in the untreated control group showed a significant (p <0.01) increase in ATP content in cerebral cortex. This rise was more pronounced in SAM P8, a learning / memory disorder model than in SAM R1, a normal animal. Therefore, the composition of the present invention improves the energy production capacity for the nervous system cells of the cerebral cortex, and the effect can be said to be greater in the pathological state than in the normal state.

On the other hand, the change in the glucose content of the cell did not show a significant difference from that in the untreated animals as shown in Table 5.

Table 5. Glucose content in cerebral cortex

Per animal animal condition (mg / dl) SAM R1 (9) Control 6.68 ± 0.18 SAM R1 (9) Experiment 6.61 ± 0.29 SAM P8 (18) Control 7.11 ± 0.20SAM P8 (16) Experiment 7.36 ± 0.25

When the result of glucose content is related to the result of change in the blood glucose of Example 7 and the increase of the ATP content, it can be seen that it is closely related to the increase in glucose utilization and its utilization rate through the cell membrane.

Example 9

Effect on protein methylase II (PM II)

The PM II activity was measured by using a slightly improved method of Gingras et al. 20 μl citrate phosphate buffer (pH 6.0), 20 μl gamma globulin (γ-globulin) (30 mg / ml) and 50 μl sample were mixed for 30 minutes at 37 ^o C, followed by 10 μl of [methyl- ³ H] SAM (Amersham) was added and reacted at 37 ^° C. for 15 minutes. To stop the reaction, 100 μl borate buffer (pH 11.0) was added to the reaction solution, and the resulting methyl ether was methylated at 37 ^° C. for 5 minutes. ⁽³ H) - was used as an organic page (organic phage) to measure the isotope amount and separated by isoamyl alcohol (isoamylalcohol) 1ml of methanol.

The results are summarized in Table 6.

The PM II activity of SAM P8, an age-promoting mouse, was 2.7 times higher than that of SAM R1, a normal animal, and a statistically significant difference was found (p <0.01). This indicates that there is a lot of abnormal protein formation and accumulation as aging progresses. The high PM II activity observed in SAM P8 was significantly reduced by administration of the composition of the present invention (p <0.01), but no change in PM II activity was observed in the normal animal SAM R1.

Table 6. Changes in activity of PM II

Animal Animal Water Condition PM II (unit.mg protein) SAM R1 (9) Control 1.55 ± 0.069 SAM R1 (9) Experiment 1.47 ± 0.046 SAM P8 (18) Control 4.20 ± 0.343 ## SAM P8 (16) Experiment 2.32 ± 0.102 **

## indicates significant difference p <0.01 for SAM R1 control, ** for SAM P8 control

Mean significant difference p <0.01.

From these results, it can be seen that the methylation of the protein acting as a substrate of PM II is increased in the cerebral cortical tissue as aging progresses, and the composition of the present invention shows a reducing effect.

<Example 10>

Histopathological observation

The inspection method is as follows. After bleeding the blood of anesthetized animals with Ethyl ether, the skull was removed without damage to the cerebrum. Brain tissues other than the cerebellum were fixed in 10% neutral formalin and used as a material for histological microscopic examination. Ethanol embedding was set to 24 hours during the treatment of the tissue to allow for a clear reading of the fine cell structure. Tissue sections were prepared with 3μm thickness and hematoxyline & eosin staining was used to observe the general characteristics of the nervous system cells.Staining was performed to observe lesions such as degeneration of axon and amyloid deposition. And PAS staining were performed.

Figure 10 shows the pathological results of SAM P8.

Necrotic neurons (Fig. 10a 40x), degenerative neurons (Fig. 10b 400x) and lipofusin deposition in the hypothalamus (Fig. 10c 400x) were observed in the cerebral cortex of animals that had not been administered Sipjeondaebotang. . Figure 10d shows the cerebral cortex and hippocampus elevation of the animal to which the composition of the present invention was administered. The deposition of eosinophilic cells in the median amygdaloid neuron of the animal to which the composition was administered was observed (Fig. 10E 200x, Fig. 10F: Magnification 400x of Fig. 10E). . From these results, the composition of the present invention seems to act directly on brain neuronal cells to improve morphological and functional degeneration.

The composition of the present invention improves the energy metabolism function using glucose (glucose) to the cerebral cortical nervous system cells, which improves the progression of aging (degeneration of nerve cells) by improving the degradation of energy metabolism function by aging or disease in nerve cells It is effective in preventing and treating degenerative cerebral nervous system diseases. In other words, the composition of the present invention improves the energy metabolism of cerebral nervous system cells, thereby improving biochemical and cellular morphological symptoms according to aging, and preventing and improving symptoms of senile dementia, vascular dementia and Alzheimer's dementia. This effect is greater in the pathological state than in the normal state, as can be seen in the changes in the ATP content of the cerebral cortex and the amount of methylated protein.

Claims (4)

Ginseng 1.6-2.4 parts by weight, Astragalus 0.7-1.3 parts by weight, Baekchul 0.7-1.3 parts by weight, Count 0.7-1.3 parts by weight, Angelica 0.7-1.3 parts by weight, Cheongung 0.7-1.3 parts by weight, Dermis 0.7-1.3 parts by weight, Licorice A prophylactic and therapeutic agent for degenerative cerebral nervous system disease, comprising 0.7-1.3 parts by weight, 0.7-1.3 parts by weight, 0.7-1.3 parts by weight and broilers 0.7-1.3 parts by weight. The method of claim 1, Prevention and treatment of senile dementia. The method of claim 1, Prevention and treatment of Alzheimer's dementia. Ginseng 1.6-2.4 parts by weight, Astragalus 0.7-1.3 parts by weight, Baekchul 0.7-1.3 parts by weight, Count 0.7-1.3 parts by weight, Angelica 0.7-1.3 parts by weight, Cheongung 0.7-1.3 parts by weight, Dermis 0.7-1.3 parts by weight, Licorice An energy metabolism improving agent of cerebral neurons comprising 0.7-1.3 parts by weight, 0.7-1.3 parts by weight and 0.7-1.3 parts by weight of broilers.